The present invention relates to mammalian tumor necrosis factor-xcex1 (TNF-xcex1) convertase enzymes. More particularly, it relates to bovine, human and other TNF-xcex1 convertases, isolated nucleic acids and recombinant vectors encoding the enzymes, methods for making the enzymes, fragments or fusion proteins thereof using recombinant DNA methodology or chemical synthesis, and to methods for using the enzymes in screening systems to identify TNF-xcex1 convertase inhibitors for the treatment of various diseases, and nucleic acids encoding a TNF-xcex1 convertase. This invention further relates to antibodies, both polyclonal and monoclonal, which specifically bind to the TNF-xcex1 convertases, and to fragments and fusion proteins of the TNF-xcex1 convertases of the invention.
TNF-xcex1, also known as cachectin, is a 17 kDa (kilodalton) protein produced by cells of the monocyte/macrophage lineage, and by other cells. A variety of biological effects, both beneficial and deleterious, have been attributed to TNF-xcex1. TNF-xcex1 is beneficial, e.g., in that it is believed to be a part of host anti-tumor defenses. It also produces detrimental effects, however, including, e.g., cardiovascular (shock, ARDS, capillary leakage syndrome), renal (nephritis, acute tubal necrosis), and gastrointestinal (ischemia, colitis, hepatic necrosis) effects, and effects on the central nervous system (fever, anorexia, altered pituitary hormone secretion). In view of the foregoing, a consensus view has developed that TNF-xcex1 is a key mediator of inflammation (including inflammatory diseases such as arthritis) and mammalian responses to injury, invasion by pathogens, and neoplasia.
The biosynthesis of human TNF-xcex1 proceeds by way of a membrane-bound precursor containing 233 amino acid residues [Wang et al., Science 228:149-154 (1985); Muller et al., Nature 335:265-267 (1987)], which is processed during cellular activation by cleavage of a 76-residue peptide to produce the mature, secreted form of TNF-xcex1. The enzyme(s) responsible for this cleavage, called TNF-xcex1 convertase, has until the present invention been elusive for most mammalian species.
A putative TNF-xcex1 convertase, called PR-3, has been isolated and cloned from human neutrophils, and it has been suggested that this enzyme can be used in screens to identify TNF-xcex1 convertase inhibitors. See International Patent Applications Publication Numbers WO 94/00555 and WO 95/24501. This enzyme, however, is not believed to be the physiologically relevant human TNF-xcex1 convertase because it is a serine protease, whereas the relevant enzyme is believed to be a metalloproteinase. Moreover, the source of the serine protease, neutrophils, is not believed to be important in the production of TNF-xcex1, and the serine protease does not cleave the precursor form of TNF-xcex1 (proTNF-xcex1) at the point expected for the physiologically relevant human enzyme.
Mohler et al. [Nature 70:218 (1994)] have partially purified another TNF-xcex1 convertase from the human monocytic cell line THP-1. This preparation, however, was very impure, and little could be said about the nature of the TNF-xcex1 convertase in the crude protein mixture of Mohler et al.
In view of the important role of TNF-xcex1 in many disease processes, there is a need for agents that can selectively block the biosynthesis of mature, secreted TNF-xcex1. The search for such agents would be greatly facilitated by the availability of substantially pure mammalian TNF-xcex1 convertases.
The present invention fills the foregoing need by providing materials and methods for identifying specific inhibitors of TNF-xcex1 convertase. More particularly, this invention provides substantially pure mammalian TNF-xcex1 convertases capable of converting proTNF-xcex1 to the mature, secreted form. This invention further provides isolated or recombinant nucleic acids encoding mammalian TNF-xcex1 convertases, and recombinant vectors and host cells comprising such nucleic acids.
This invention further provides a method for making a mammalian TNF-xcex1 convertase, comprising culturing a host cell comprising a nucleic acid encoding a mammalian TNF-xcex1 convertase under conditions in which the nucleic acid is expressed. In some embodiments, the method further comprises isolation of the TNF-xcex1 convertase from the culture.
This invention also provides polypeptides comprising a fragment of a TNF-xcex1 convertase having an amino acid sequence corresponding to the sequence of at least about 8 contiguous residues of the complete enzyme sequence. Preferably, the polypeptides comprise at least about 12, more preferably at least about 20, and most preferably at least about 30 such residues.
Still further, this invention provides fusion proteins comprising a TNF-xcex1 convertase or a polypeptide thereof covalently linked to a fusion partner.
The present invention also provides antibodies, both polyclonal and monoclonal, that specifically bind to one or more of the TNF-xcex1 convertases or to a polypeptide thereof. Also provided are anti-idiotypic antibodies, both monoclonal and polyclonal, which specifically bind to the foregoing antibodies.
This invention still further provides a method of treatment comprising administering to a mammal afflicted with a medical condition caused or mediated by TNF-xcex1, an effective amount of an antibody, or an antigen-binding fragment thereof, that specifically binds to a mammalian TNF-xcex1 convertase, and pharmaceutical compositions comprising such antibodies or fragments and pharmaceutically acceptable carriers.
The present invention also provides a method for identifying an inhibitor of a mammalian TNF-xcex1 convertase, comprising:
(a) contacting a mammalian TNF-xcex1 convertase in the presence of substrate with a sample to be tested for the presence of an inhibitor of the convertase; and
(b) measuring the rate of cleavage of the substrate;
whereby an inhibitor of the TNF-xcex1 convertase in the sample is identified by measuring substantially reduced cleavage of the substrate, compared to what would be measured in the absence of such inhibitor.
In a preferred embodiment, the contacting of the convertase with the sample in the presence of substrate occurs on the surface of a mammalian host cell comprising one or more nucleic acids encoding a mammalian TNF-xcex1 convertase and a substrate of the convertase.